Purification of microbial cell fermentation homogenates

Abstract

A method for purifying a desired heterologous polypeptide from microbial fermentation broth or homogenate in which it is produced and solubilized is described. This method involves adding to the broth or homogenate an effective amount of a solution of 6,9-diamino-2-ethoxyacridine lactate (ethacridine lactate) to precipitate host cell impurities under conditions wherein the majority of polypeptide remains soluble, and separating the desired polypeptide from the broth or homogenate. The broth or homogenate containing the ethacridine lactate and polypeptide is also disclosed.

1. A microbial cell fermentation broth or homogenate comprising ethacridine lactate of greater than 0.1% (weight/volume) and a polypeptide heterologous to the cells, wherein the pl of the polypeptide is greater than the average pl of the host proteins contained in the cells, wherein the broth or homogenate is at a temperature of from about 50 to 65° C., and wherein the polypeptide is an antibody or antibody fragment. 
     
     
       2. The broth or homogenate of  claim 1  that is from bacterial cells. 
     
     
       3. The broth or homogenate of  claim 1  that is from  E. coil.    
     
     
       4. The broth or homogenate of  claim 1  wherein the polypeptide is a recombinant polypeptide. 
     
     
       5. The broth or homogenate of  claim 1  wherein the polypeptide is F(ab′) 2 . 
     
     
       6. The broth or homogenate of  claim 1  wherein the polypeptide is anti-IgE, anti-CD18, anti-VEGF, anti-tissue factor, 2C4, anti-Her-2, anti-CD20, anti-CD40, or anti-CD11a antibody. 
     
     
       7. The broth or homogenate of  claim 1  wherein the polypeptide is anti-CD18F(ab′) 2 , anti-tissue factor F(ab′) 2 , full-length anti-tissue factor antibody, or anti-VEGF antibody. 
     
     
       8. The broth or homogenate of  claim 1  wherein the polypeptide is soluble in the broth or homogenate. 
     
     
       9. The broth or homogenate of  claim 1 , wherein the concentration of ethacridine lactate is about 0.1-5%. 
     
     
       10. The broth or homogenate of  claim 9 , wherein the concentration of ethacridine lactate is about 0.4-5%. 
     
     
       11. The broth or homogenate of  claim 10 , wherein the concentration of ethacridine lactate is about 0.6-5%. 
     
     
       12. The broth or homogenate of  claim 1 , wherein the conductivity is less than 16 milliSiemens (mS/cm). 
     
     
       13. The broth or homogenate of  claim 12 , wherein the conductivity is about 1-15 mS/cm. 
     
     
       14. The broth or homogenate of  claim 13 , wherein the conductivity is about 1-10 mS/cm. 
     
     
       15. The broth or homogenate of  claim 14 , wherein the conductivity is about 1-5 mS/cm. 
     
     
       16. The broth or homogenate of  claim 1 , wherein the pH ranges from about 4-10. 
     
     
       17. The broth or homogenate of  claim 16 , wherein the pH is about 4-9. 
     
     
       18. The broth or homogenate of  claim 17 , wherein the pH is about 5-9. 
     
     
       19. The broth or homogenate of  claim 18 , wherein the pH is about 6 -9. 
     
     
       20. The broth or homogenate of  claim 19 , wherein the pH is about 6-7.